5.1 Strand Invasion Based Amplification (SIBA®)
5.1.1 SIBA® reaction
SIBA® reaction is not completely understood. In this chapter is theory about SIBA®
reaction present. At the beginning all ssDNA structures are coated with gp32 binding proteins. UvsX reads seeding area of IO and displaces bound gp32 molecules. The IO invades the complementary region of the target duplex of the template which results in partial separation if the duplex strands. The partially separated ssDNA strand is coated with gp32. Gp32 molecules are released when primers are bound to the peripheral region of the IO that are dissociated.
Polymerase with displacement activity is able to extend the dissociated duplex template from primers. The forward primer displaces the IO during extension of the target template and when 2’-O-methyl RNA is reached the Bsu polymerase and IO are released. Each SIBA® cycle generates two copies of the target duplex (figure 3).
[133]
Figure 3. Steps of the SIBA® reaction. [133]
37 5.1.2 IO-quenching technique
The IO-quenching detection technique makes possible to use multiple amplicons in the same SIBA® reaction as well it makes SIBA® more resistant to non-specific amplification. The technology has also been found to be highly sensitive, but theory behind this is unclear. IO has on its 3’ end a quencher molecule while the 5’ end of the RV primer is fluorophore. If a specific target sequence is not present in the sample, primers or IO cannot anneal with the target sequence which causes detection of a constant signal level. Whereas, if a detectable amount of the target sequence is present in the sample, primers and IO bind and cause the signal level to drop when IO’s quencher is quenching the signal of the fluorophore. The more amplicons are synthetized the more the signal level drops.
5.1.3 Reagents
The components used in 20 µl SIBA® reaction are presented below. Commonly, the SIBA® reaction consists of 140 ng/µl UvsX, 200 µM dNTPs, 0.0625 U/µl Bsu, 250 ng/µl gp32, 0.025 U/ml creatine phosphokinase, 0.0125 U/µl sucrose phosphorylase, 0.5 mM EDTA, 4 mM DTT, 0.1 mg/ml BSA, 5 % DMSO, 2mM ATP, 5 % PEG400, 10 mM magnesium acetate, 60 mM Tris-phosphocreatine, 0.1x SYBR®
Green I, 10 mM Tris-Acetate (pH 8.0) and 200 nM each oligo. [133]
UvsX
UvsX is the bacteriophage T4 analog of E. coli RecA protein, which binds cooperatively to a single strand of a double stranded DNA template. Uvsx catalyzes paring and strand exchange between the ssDNA and the complementary strand of the duplex, creating a displacement loop (D-loop). Enzymes binds DNA regions with lengths of 30-40 nt and reactions are ATP-dependent. UvsX activity is assisted by the UvsY protein. In SIBA®, UvsY is not present being replaced with higher amount
38 of UvsX. UvsX is ATP-dependent and thus in SIBA®, an ATP regeneration system is needed. [133, 138]
Bsu
Bacillus subtilis DNA polymerase I Large Fragment (Bsu) recognizes the 5’ end of the primer, binds to the template and generates DNA sequence from dNTPs. Bsu lacks the exonuclease domain. Bsu has strand displacement activity, which is important for detachment of the IO. [115, 139]
Gp32
The T4 helix-destabilizing protein, gene 32 protein plays a role in amplification by binding single-stranded regions of the template, IO and primers and is required throughout DNA polymerization. By binding to template strands at the replication fork, gp32 may effect local unwinding, assists in the removal of the secondary structure in ssDNA, allowing for the complete assembly of uvsX along the ssDNA, and protects the single stands against nucleases [140]. Unlike an enzyme, the gp32 is a reagent that changes the rate and the equilibrium of the reaction. High salt concentration and levels of Mg2+ in the reaction favors dsDNA renaturation by gp32 rather than denaturation before replication starts. Gp32 is recycled after binding single stranded regions of the template facilitating replication. [141]
Creatine phosphokinase
Creatine phosphokinase (CPK) is responsible for catalyzing reversible reaction of ATP hydrolysis / regeneration. An ATP regeneration system is needed because of ATP-dependent UvsX. Reactions catalyzed by CPK are:
Forward reaction: ADP + phosphocreatine ATP + creatine (1)
39 Reverse reaction: ATP + creatine phosphocreatine + ADP (2)
CPK requires Ca2+ or Mg2+ for its activity. Excess amounts of Ca2+ or Mg2+ are inhibitory for forward reaction and alters the equilibrium of reverse reaction.
Presence of thiol groups e.g. cysteine or dithiothreitol has been found to increase activity of the CPK. Optimal pH is different for forward and reverse reaction; optimal pH for forward reaction is 7.2. [142, 143]
Phosphocreatine
Phosphocreatine is a substrate for CPK and needed in ATP regeneration system.
Sucrose phosphorylase
Sucrose phosphorylase is important enzyme of energy metabolism which catalyzes a number of glucosal transfer reactions. SIBA® utilizes the reaction converting sucrose to D-fructose and α-D-glucose-1-phosphate.
Reaction: Sucrose + Pi α-D-glucose-1-phosphate + D-fructose (3)
In the SIBA® reaction sucrose phosphorylase plays a role in binding of inorganic phosphates (Pi). The accumulation of Pi is able to chelate magnesium and would thus inhibit enzymes needed in amplification. The sucrose phosphorylase reaction has been added to prevent this inhibition. [115]
dNTPs (A, C, G, U)
dNTPs are single nucleotides used as starting material of the DNA polymerization. In the SIBA® reaction, thymine (T) normally used in the DNA synthesis, is replaced with
40 uracil (U) so that the uracil-DNA glycosylase decontamination method could be used if needed. In the reaction mix the uracil concentration is four times larger than that of the other nucleotides. It has been postulated that this replaces the lack of dTTP in the reaction.
Sucrose
Sucrose is a substrate for sucrose phosphorylase.
EDTA
Ethylenediaminetetraacetic acid (EDTA) is a chelate forming ligand that chelates most metal-ions in the molar ratio 1:1. [144] Specifically it chelates divalent cations, such as Mg2+ which is used in the SIBA® reaction as a cofactor of the enzymes. EDTA is used in the SIBA® reaction mix to prevent premature action of the enzymes during storage. [145] EDTA is also present in the Tris-EDTA (TE) buffer (0.5 mM) which is used as a diluent of the oligos. In this master’s thesis also templates (synthetic template and extracted DNA from Yersinia strains) were diluted in TE-buffer for protecting templates from nucleases.
DTT
Dithiothreitol (DTT) is a commonly used sulfhydryl reducing agent in biochemistry along with 2-mercaptoethanol. The mechanism of disulfide reduction by thiols is an exchange of the thiolate anion. DTT prevents enzymes from oxidation of sulfhydryl which can lead inhibition of the enzyme. DTT is highly stable reducing agent for long-term storage of proteins when metal chelates such as EDTA are present. [146]
DTT is used in the reaction mix to protect enzymes from oxidation.
41 BSA
Bovine serum albumin (BSA) has been used widely for relieving interference in PCR and other enzymatic reactions. It has relieved inhibition in case of substances with phenolic groups which are known to bind to proteins by forming hydrogen bonds with peptide bond oxygen. BSA is also known to bind with lipids via hydrophobic forces and anions. Therefore BSA may prevent binding of a variety of interfering substances and inactivation of DNA polymerase used in amplification reactions.
[147] BSA also increases total protein concentration in the reaction which might be relevant for action of the enzymes.
DMSO
Dimethyl sulfoxide (DMSO) prevents formation of secondary structure in the DNA template and primers, and decreases melting temperature of the reaction. It interferes with the self-complementarity of the DNA minimizing interfering reactions. [148] Especially DMSO facilitates amplification of supercoiled plasmids or DNA templates with high GC-content [149].
ATP
Adenosine triphosphate (ATP) is an energy-bearing molecule used by enzymes. ATP regulates the conformational changes required for enzyme action through binding and hydrolysis processes. [150] ATP-dependent enzymes such as UvsX hydrolyze ATP to ADP and inorganic phosphate during catalysis of the specific reactions. [151]
This initial amount of ATP is not enough for the whole SIBA® reaction and ATP needs to be regenerated. [115]
42 PEG400
Poly (ethylene glycol) (PEG) is a linear polymer molecule consists of ethylene glycol molecules. PEG400 is a low molecular weight PEG molecule (Mw around 400 g/mol) which is used in the SIBA® reaction as a crowding agent bringing other essential reactants close to each other. [152]
Magnesium acetate
Magnesium cation (Mg2+) is an important cofactor for enzymes. It performs structural and catalytic functions which result in catalytic activity of the enzymes.
Also DNA and RNA binds efficiently Mg2+ as it participates in neutralization of the polyanionic charge of the nucleic acid. [150, 153]
Tris-Phosphocreatine
Phosphocreatine is a source of Pi. As earlier mentioned, creatine phosphokinase needs phosphocreatine to generate ATP from ADP. Phosphocreatine stored in tris buffer is used in SIBA®.
SYBR® Green I
SYBR® Green I is sensitive stain for detecting any dsDNA present in the reaction.
Wave length of absorption/emission are 497/520 nm. SYBR Green I is used in SIBA®
when IO-quenching is not used or otherwise total amplification of dsDNA needs to be detected. [154]
Tris Acetate (pH 8.0)
Tris acetate buffer is used for pH control of the reaction.
43 Oligos
In this master’s thesis all oligos (FW, RV and IO) are prepared in TE-buffer.
5.2 Loop-mediated isothermal amplification (LAMP)
Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification method developed by Notomi et al. 1999. [123] Several hundreds of assays for viruses, bacteria, fungi, protista and mammalian DNA, have been developed and published [155]. The first described LAMP method used DNA polymerase with high strand displacement activity and four primers that recognized six distinct regions on the target DNA [156, 157]. Soon after this Nagamine at al.
2001 described an improved LAMP-method using additional two loop-primers which accomplish an accelerated reaction [158]. Primers used in this improved LAMP method are FIP (forward inner primer), BIP (backward inner primer), F3, B3 and loop F and loop B [156, 158]. FIB and BIF both contain two distinct sequences corresponding to the sense and antisense sequences of the target DNA separated by a spacer sequence. Two outer primers called F3 and B3 are complementary sequences for F3c and F3c areas of target DNA. LAMP is also capable of amplifying RNA molecules in a single-tube reaction when reverse transcriptase (RTase) is used together with DNA polymerase [156].
The LAMP method can be divided in three stages including starting material producing step, cycling amplification step and elongation and recycling step.
Reaction stages are described in figure 4.
In the initial stage starting material so called dumbbell-like DNA form is produced.
In the first step FIP anneals to target ssDNA and complementary DNA strain is generated by Bst DNA polymerase (figure 4 step 1). In the second step F3 anneals and during elongation displaces FIP-linked complementary strand which has formed
44 stem-loop structure at its 5’ end (figure 4 steps 2 and 3). This FIP-linked strand plays a role as a template for BIP and B3 primers, respectively (figure 4 steps 4-6). The final product of these steps is a structure with stem-loops at 5’ end and 3’ end.
The starting material producing stage is followed by cycling amplification stage in which only FIP and BIP primers play a role. FIP anneals to 3’-loop of the starting material and elongation of FIP and F1 (starting material structures looped 3’-end) starts. After annealing the original 5’-end loop opens and new loop-structure is generated. Elongation of this B1 and BIP proceeds, respectively (figure 4 steps 8-11). [156]
After the cycling amplification step the elongation and recycling step starts generated by FIP or BIP. If loop primers are used in the LAMP reaction they are annealed and elongated in this stage. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primer, and prime strand displacement DNA synthesis (figure 4 steps 12-20). [157, 158]
The LAMP reaction can be carried out in a total 25 µl mixture that contains 0.8 µM of each FIP and BIP, 0.2 µM of each F3 and B3, 0.4 µM of each loop primers F and B, 1 M betaine, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM MgSO4, 0.1 % Triton X-100, 8 Units of Bst DNA polymerase large fragment, 400 µM (if loop primers are not used) or 1.6 mM (if loop primers are used) of each dNTP and aspecified amount of target DNA. 0.25 µg/ml ethidium bromide can be added when end-point detection method is used. [156-158]
The reaction mixture can be first denatured at 95 °C for 5 min (template denaturation) and chilled before adding Bst DNA polymerase but this step is not,
45 however, required. Heat denaturation can facilitate amplification loosing affinity between DNA strands. The LAMP reaction can be performed at 60-65 °C for 1 hour.
[158] Detection time of different analytes can be only few minutes depending on amount of template copies. The yield of the LAMP product using loop primers is at least 500 µg/ml [157]. The LAMP method is protected by a PCT-patent. [123]
Figure 4. Steps of LAMP reaction with four primers. [156]
46 5.3 Recombinase polymerase amplification (RPA)
Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique developed by Piepenburg et al. in 2002 [129]. Isothermal amplification is achieved by several enzymes and the binding of opposing nucleotide primers to template. Enzymes required for RPA reaction are recombinases uvsX, uvsY and gp32, Bsu DNA polymerase large fragment and enzymes of ATP generating system phosphocreatine and creatine kinase. [134] RPA is also capable of detecting RNA molecules if RTase is added to the reaction [159].
Reaction starts with binding of formed recombinase-primer complexes to the target DNA sequences in different strands (sense and antisense). Template denaturation is not required because recombinase-primer complex can scan dsDNA and thus facilitates strand displacement. The Gp32 single-stranded DNA binding proteins available in the reaction mixture stabilize the D-loop by binding to displaced single strands and thus preventing dissociation of the primers. When DNA polymerases cross, parental DNA strains are separated, and extension will continue until two identical dsDNA molecules have been synthesized. A schematic illustration of the steps of the PRA is shown in figure 5. [134]
In total a 20 µl reaction mixture can contain 50mM Tris (pH 7.9), 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM DTT, 5% Carbowax20M, 200 µM dNTPs, 3 mM ATP, 50 mM phosphocreatine, 100 ng/µl creatine kinase, 30 ng/µl Bsu and 900 ng/µl gp32, 120 ng/µl uvsX and 30 ng/µl uvsY. Primer concentration varies according to assay. If a multiplex assay is performed, Carbowax20M concentration increases 5.5 %. [134]
UvsX is ATP-dependent enzyme and in the presence of ATP, uvsX binds to dsDNA whereas ATP hydrolysis permits separation of the uvsX from dsDNA molecules and
47 thus allows gp32 binding to ssDNA [134, 160]. Hence an ATP generating system is needed in the reaction. A PEG called Carbowax20M is used in the reaction to establish favorable reaction conditions for RPA reaction bringing reaction compounds close to each other. [134]
The RPA reaction does not require high or precise temperature and it can proceed at temperatures between 25 °C and 42 °C. However, many assays are performed at the temperature of 39 °C because it is close to optimal temperature of the polymerase enzyme which is 37 °C [161].
RPA is highly sensitive and can detect 10 copies of template in less than 40 minutes and even two copies have been detected but in this case amplification is slower.
Specific detection of the RPA product is achieved by using specially designed probes which recognize the complementary region of RPA amplicon. [134] High specificity has been achieved for different assays for example Mycobacterium tuberculosis [161]. The method has PCT-patent granted in 2002 [129]. Also the multiplexing method has been patented in 2012 [162].
Figure 5: Steps of the RPA cycle.
48 5.4 Strand-displacement amplification (SDA)
Strand-displacement amplification (SDA) is an isothermal amplification technique developed by Becton Dickinson. It is used primarily for amplification of DNA, but can amplify RNA by incorporating an initial stage of reverse transcription. [163] The technique is based on the ability of a DNA polymerase lacking exonuclease activities to extend the 3' end at the nick and displace the downstream strand, and the ability of a restriction enzyme HincII or BsoBI to nick the unmodified strand of a hemiphosphorothioate form of its recognition site in dsDNA. Exponential target DNA amplification is achieved by coupling sense and antisense reactions. Strands displaced from a sense reaction are used as a template of the antisense reaction and vice versa. 107-fold amplification can be achieved by SDA in a few hours. [121, 164, 165]
Steps of the SDA reaction are described in figure 6. Initially ssDNA fragments serve as a target and bind to an SDA primer containing a recognition sequence for HincII.
DNA replication uses dCTP, dGTP, TTP, and dATP alpha S for producing a double-stranded hemiphosphorothioate recognition site. After extension HincII nicks the unprotected primer strand at its recognition site. Then DNA polymerase extends the 3' end at the nick and displaces the downstream fragment. The polymerization step regenerates a nickable recognition site. Nicking and polymerization/displacement steps cycle produces single-stranded complementary copies of the target fragment.
[164]
Restriction enzyme digestion is performed before SDA. RsaI is used to digest sample DNA to fragments, which serves as a target. RsaI cleavage is performed using 10 U/µg of DNA in 50 mM Tris-HCl, pH 8 or in 10 mM MgCl2 for 1 hour at 37 °C followed by 2 min incubation at 95 °C. [164] SDA reaction is performed in 37-40 °C.
The first described SDA reaction was done in 100 µl and the mix contained target
49 DNA in a solution of 100 units of HinclI, 2.5 units of E. coli DNA polymerase I (exo- Klenow), 1 mM dGTP, 1 mM dCTP, 1 mM TTP, and 1 mM dATP alpha S, 50 mM Tris HCI (pH 7.4), 6 mM MgCl2, 50 mM NaCl, 50 mM KCI, 1 % glycerol, and 1 µM primers including recognition sequence of the HincII. DATP alpha S has a role as a protector of the sequence due restricting formed DNA strand with HindII. Samples were incubated in 95 °C for 4 min to denature the target fragment followed by 4 min at 37 °C to anneal primers before addition HincII and DNA polymerase to the reaction.
Upon addition of HincIl and DNA polymerase, amplification reaction mixtures were incubated 1 to 5 h at selected temperature. [164] Improved SDA reaction types have also been described, using four primers instead of two and replacing NaCl and KCl with KiPO4 (pH 7.4), MgCl2 with magnesium acetate and adding organic solvent 1-methyl-2-pyrrolidinone. [165]
Figure 6. Steps of the SDA cycle. [164]
50 5.5 Self-sustained sequence replication (3SR)
Self-sustained sequence replication (3SR) is an isothermal method for nucleic acid amplification developed by Fahy et al. 1990 [118, 166]. The method was first developed for RNA but it has been applied for DNA amplification also. Application of the 3SR system to DNA target sequences requires the use of thermal denaturation during the initial synthesis of cDNA containing the T7 promoter sequence. If thermal denaturation steps are not done, the duplex DNA cannot serve as a substrate for the 3SR reaction. [167] The 3SR is performed at a temperature of 37-42 °C and it relies on two primers (A and B) and the activity of three enzymes. Each primer contains the T7 RNA polymerase binding sequence and the transcriptional initiation site. The remaining sequence is complementary to the target sequence. Enzymes used in the reaction are avian myeloblastosis virus reverse transcriptase (AMV-RT), ribonuclease H (RNase H) and T7 RNA polymerase. T7 RNA polymerase is able to amplify target RNA by producing multiple copies of target RNA using a double-stranded DNA generated by AMV-RT. [168]
Components of the first described 3SR reaction in 100 µl reaction were: the target RNA, 40 mM Tris-HCI (pH 8.1), 20 mM MgCl2, 25 mM NaCl, 2 mM spermidine hydrochloride, 5 mM dithiothreitol, bovine serum albumin (80 µg/ml), 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 250 ng of both primers, 30 units of AMV reverse transcriptase, 100 units of T7 RNA polymerase, and 4 units of E. coli RNase H. Reaction is preheated in 65 °C for 1 minute and cooled in 37 °C for 2 minutes before enzymes are added. [169]
The 3SR reaction starts from annealing of the A primer (which has a T7 promoter sequence) to sense RNA strand of the target (figure 7, step 1). AMV-RT transcribes a strand of antisense DNA, followed by digestion of the sense RNA strand by RNase H (figure 7, step 2 and 3). B primer anneals to the antisense ssDNA followed by
51 synthesis of a complementary strand of DNA by AMV-RT (figure 7, steps 4-6). [169]
Generated cDNAs are used to produce multiple copies of antisense RNA transcript
Generated cDNAs are used to produce multiple copies of antisense RNA transcript